Hsp70 Knockdown in the Brine Shrimp Artemia franciscana: Implication on Reproduction, Immune Response and Embryonic Cuticular Structure.

The potential functional role(s) of heat shock protein 70 (Hsp70) in the brine shrimp, Artemia franciscana, a crucial crustacean species for aquaculture and stress response studies, was investigated in this study. Though we have previously reported that Hsp70 knockdown may have little or no impact on Artemia development, the gestational survival and number of offspring released by adult females were impaired by obscuring Hsp70 synthesis. Transcriptomic analysis revealed that several cuticle and chitin synthetic genes were downregulated, and carbohydrate metabolic genes were differentially expressed in Hsp70-knockdown individuals. A more comprehensive microscopic examination performed in this study revealed exoskeleton structural destruction and abnormal eye lenses featured in Hsp70-deficient adult females 48 h after Hsp70 dsRNA injection. Cysts produced by these Hsp70-deficient broods, instead, had a defective shell and were smaller in size, whereas nauplii had shorter first antennae and a rougher body epicuticle surface. Changes in carbohydrate metabolism caused by Hsp70 knockdown affected glycogen levels in adult Artemia females, as well as trehalose in cysts released from these broods, indicating that Hsp70 may play a role in energy storage preservation. Outcomes from this work provided novel insights into the roles of Hsp70 in Artemia reproduction performance, cyst formation, and exoskeleton structure preservation. The findings also support our previous observation that Hsp70 knockdown reduced Artemia nauplius tolerance to bacterial pathogens, which could be explained by the fact that loss of Hsp70 downregulated several Toll receptor genes (NT1 and Spaetzle) and reduced the integrity of the exoskeleton, allowing pathogens to enter and cause infection, ultimately resulting in mortality.

Seagrass genomes reveal ancient polyploidy and adaptations to the marine environment.

We present chromosome-level genome assemblies from representative species of three independently evolved seagrass lineages: Posidonia oceanica, Cymodocea nodosa, Thalassia testudinum and Zostera marina. We also include a draft genome of Potamogeton acutifolius, belonging to a freshwater sister lineage to Zosteraceae. All seagrass species share an ancient whole-genome triplication, while additional whole-genome duplications were uncovered for C. nodosa, Z. marina and P. acutifolius. Comparative analysis of selected gene families suggests that the transition from submerged-freshwater to submerged-marine environments mainly involved fine-tuning of multiple processes (such as osmoregulation, salinity, light capture, carbon acquisition and temperature) that all had to happen in parallel, probably explaining why adaptation to a marine lifestyle has been exceedingly rare. Major gene losses related to stomata, volatiles, defence and lignification are probably a consequence of the return to the sea rather than the cause of it. These new genomes will accelerate functional studies and solutions, as continuing losses of the 'savannahs of the sea' are of major concern in times of climate change and loss of biodiversity.

The effects of Pandanus tectorius leaf extract on the resistance of White-leg shrimp Penaeus vannamei towards pathogenic Vibrio parahaemolyticus.

Pandanus tectorius leaf extract effect on the White-leg shrimp Penaeus vannamei tolerance against Vibrio parahaemolyticus were investigated in this study. Thirty shrimp post-larvae measured at approximately 1 cm were exposed for 24 h to 0.5, 1, 2, 3, 4, 5 and 6 g/L leaf extract and subsequently observed for survival and immune-related genes expression (Hsp70, ProPO, peroxinectin, penaeidin, crustin and transglutaminase), followed by determination of their tolerance and histological tissue profiles upon Vibrio challenge. Survival of shrimps treated with 6 g/L of leaf extract improved by up to 95% to controls. Hsp70, crustin, and prophenoloxidase mRNA levels were observed to be 8.5, 10.4, and 1.5-fold higher, respectively. Histopathological analysis of the hepatopancreas and the muscle tissues revealed major tissue degeneration in Vibrio-challenged shrimps but not in shrimps primed with P. tectorius leaf extract. Of all the dose examined, the best pathogen resistance results were obtained with a 24 h incubation of shrimp in 6 g/L P. tectorius methanolic leaf extract. The tolerance towards V. parahaemolyticus might be associated with the increased regulation of Hsp70, prophenoloxidase and crustin upon exposure to the extract, all immune-related proteins essential for pathogen elimination in Penaeid shrimp. The present study primarily demonstrated that P. tectorius leaf extract is a viable alternative for enhancing P. vannamei post-larvae resistance against V. parahaemolyticus, a major bacterial pathogen in aquaculture.

Cenozoic colonisation of the Indian Ocean region by the Australian freshwater-originating glassperch family Ambassidae (Teleostei).

We examined the phylogeny and biogeography of the glassperch family Ambassidae (Teleostei), which is widely distributed in the freshwater, brackish and marine coastal habitats across the Indo-West Pacific region. We first built a comprehensive time-calibrated phylogeny of Ambassidae using five genes. We then used this tree to reconstruct the evolution of the salinity preference and ancestral areas. Our results indicate that the two largest genera of Ambassidae, Ambassis and Parambassis, are each not monophyletic. The most recent common ancestor of Ambassidae was freshwater adapted and lived in Australia about 56 million years ago. Three independent freshwater-to-marine transitions are inferred, but no marine-to-freshwater ones. To explain the distribution of ambassids, we hypothesise two long-distance marine dispersal events from Australia. A first event was towards Southeast Asia during the early Cenozoic, followed by a second one towards Africa during mid-Cenozoic. The phylogenetic signal associated with the salinity adaptation of these events was not detected, possibly because of the selective extinction of intermediate marine lineages. The Ambassidae shares two characteristics with other freshwater fish groups distributed in continental regions surrounding the Indian Ocean: They are too young to support the hypothesis that their distribution is the result of the fragmentation of Gondwana, but they did not retain the phylogenetic signal of their marine dispersal.

Population Genetic Structure of Marine Leech, Pterobdella arugamensis in Indo-West Pacific Region.

Grouper aquaculture is rapidly expanding in both tropical and subtropical regions. The presence of marine leeches (Pterobdella arugamensis; previously named Zeylanicobdella arugamensis) infesting cultured groupers, however, can have a fatal effect on grouper aquaculture production and cause significant economic loss. Understanding the marine leech's population structure is therefore important to determine its possible distributional origin and distributional mechanisms, which will help monitor and mitigate the infestation. In this study, a total of 84 marine leeches collected from cultured hybrid groupers Epinephelus spp. in Brunei Darussalam, Malaysia and Indonesia were identified as P. arugamensis, based on morphological and mitochondrial cytochrome c oxidase subunit I gene sequence analyses. These leech samples, together with additional sequences from the GenBank database, were grouped into four genetically distinct haplogroups: (1) Asia Pacific, (2) Borneo, (3) Surabaya and (4) Iran. The four populations were found to be highly diverged from each other. The results also suggested that the samples from the Asia Pacific population could be dispersed and transported from Indonesia.

Genetic diversity, population structure and historical demography of the two-spined yellowtail stargazer (Uranoscopus cognatus).

Benthic species, though ecologically important, are vulnerable to genetic loss and population size reduction due to impacts from fishing trawls. An assessment of genetic diversity and population structure is therefore needed to assist in a resource management program. To address this issue, the two-spined yellowtail stargazer (Uranoscopus cognatus) was collected within selected locations in the Indo-West Pacific (IWP). The partial mitochondrial DNA cytochrome c oxidase subunit 1 and the nuclear DNA recombination activating gene 1 were sequenced. Genetic diversity analyses revealed that the populations were moderately to highly diversified (haplotype diversity, H = 0.490-0.900, nucleotide diversity, π = 0.0010-0.0034) except sampling station (ST) 1 and 14. The low diversity level, however was apparent only in the matrilineal marker (H = 0.118-0.216; π = 0.0004-0.0008), possibly due to stochastic factors or anthropogenic stressors. Population structure analyses revealed a retention of ancestral polymorphism that was likely due to incomplete lineage sorting in U. cognatus, and prolonged vicariance by the Indo-Pacific Barrier has partitioned them into separate stock units. Population segregation was also shown by the phenotypic divergence in allopatric populations, regarding the premaxillary protrusion, which is possibly associated with the mechanism for upper jaw movement in biomechanical feeding approaches. The moderate genetic diversity estimated for each region, in addition to past population expansion events, indicated that U. cognatus within the IWP was still healthy and abundant (except in ST1 and 14), and two stock units were identified to be subjected to a specific resource management program.

Pandanus tectorius fruit extract promotes Hsp70 accumulation, immune-related genes expression and Vibrio parahaemolyticus tolerance in the white-leg shrimp Penaeus vannamei.

Plants and herbal extracts are indispensable for controlling the spread of disease-causing bacteria, including those that infect aquatic organisms used in aquaculture. The use of plant or herbal extract is expected to be safe for aquatic animals and less harmful to the environment, as opposed to conventional therapeutic alternatives such as antibiotics that promote the occurrence of potential antibiotic-resistant bacteria when used improperly. The efficacy of Pandanus tectorius fruit extract in the regulation of Hsp70 expression, pro-phenoloxidase (ProPO), peroxinectin, penaeidin, crustin and transglutaminase, all immune peptides essential for Vibrio tolerance in white leg shrimp, Penaeus vannamei, was investigated in this study, which included the determination of the safety levels of the extract. Tolerance of shrimp against Vibrio parahaemolyticus, a pathogenic bacteria that causes Acute Hepatopancreas Necrosis Disease (AHPND), was assessed on the basis of median lethal dose challenge survival (LD50 = 106 cells/ml). Mortality was not observed 24 h after exposure of 0.5-6 g/L of the fruit extract, indicating that P. tectorius was not toxic to shrimp at these concentrations. A 24-h incubation of 2-6 g/L of the fruit extract increased shrimp tolerance to V. parahaemolyticus, with survival doubled when the maximum dose tested in this study was used. Concomitant with a rise in survival was the increase in immune-related proteins, with Hsp70, ProPO, peroxinectin, penaeidin, crustin and transglutaminase increased 10, 11, 11, 0.4, 8 and 13-fold respectively. Histological examination of the hepatopancreas and muscle tissues of Vibrio-infected shrimp primed with P. tectorius extract revealed reduced signs of histopathological degeneration, possibly due to the accumulation of Hsp70, a molecular chaperone crucial to cellular protein folding, tissue repair and immune response of living organisms, including Penaeid shrimp.

Recent population expansion of longtail tuna Thunnus tonggol (Bleeker, 1851) inferred from the mitochondrial DNA markers.

The population genetic diversity and demographic history of the longtail tuna Thunnus tonggol in Malaysian waters was investigated using mitochondrial DNA D-loop and NADH dehydrogenase subunit 5 (ND5). A total of 203 (D-loop) and 208 (ND5) individuals of T. tonggol were sampled from 11 localities around the Malaysian coastal waters. Low genetic differentiation between populations was found, possibly due to the past demographic history, dispersal potential during egg and larval stages, seasonal migration in adults, and lack of geographical barriers. The gene trees, constructed based on the maximum likelihood method, revealed a single panmictic population with unsupported internal clades, indicating an absence of structure among the populations studied. Analysis on population pairwise comparison ФST suggested the absence of limited gene flow among study sites. Taken all together, high haplotype diversity (D-loop = 0.989-1.000; ND5 = 0.848-0.965), coupled with a low level of nucleotide diversity (D-loop = 0.019-0.025; ND5 = 0.0017-0.003), "star-like" haplotype network, and unimodal mismatch distribution, suggests a recent population expansion for populations of T. tonggol in Malaysia. Furthermore, neutrality and goodness of fit tests supported the signature of a relatively recent population expansion during the Pleistocene epoch. To provide additional insight into the phylogeographic pattern of the species within the Indo-Pacific Ocean, we included haplotypes from GenBank and a few samples from Taiwan. Preliminary analyses suggest a more complex genetic demarcation of the species than an explicit Indian Ocean versus Pacific Ocean delineation.

Cyst viability and stress tolerance upon heat shock protein 70 knockdown in the brine shrimp Artemia franciscana.

Females of the brine shrimp Artemia franciscana produce either free-swimming nauplii via ovoviviparous pathway of reproduction or encysted embryos, known as cysts, via oviparous pathway, in which biological processes are arrested. While previous study has shown a crucial role of ATP-dependent molecular chaperone, heat shock protein 70 (Hsp70) in protecting A. franciscana nauplii against various abiotic and abiotic stressors, the function of this protein in diapausing embryos and cyst development, however, remains unknown. RNA interference (RNAi) was applied in this study to examine the role of Hsp70 in cyst development and stress tolerance, with the latter performed by desiccation and freezing, a common method used for diapause termination in Artemia cysts. Hsp70 knockdown was apparent in cysts released from females that were injected with Hsp70 dsRNA. The loss of Hsp70 affected neither the development nor morphology of the cysts. The time between fertilization and cyst release from Artemia females injected with Hsp70 dsRNA was delayed slightly, but the differences were not significant when compared to the controls. However, the hatching percentage of cysts which lacks Hsp70 were reduced following desiccation and freezing. Taken together, these results indicated that Hsp70 possibly plays a role in the stress tolerance but not in the development of diapause-destined embryos of Artemia. This research makes fundamental contributions to our understanding of the role molecular chaperone Hsp70 plays in Artemia, an excellent model organism for diapause studies of the crustaceans.

Applications of Next-Generation Sequencing Technologies and Computational Tools in Molecular Evolution and Aquatic Animals Conservation Studies: A Short Review.

Aquatic ecosystems that form major biodiversity hotspots are critically threatened due to environmental and anthropogenic stressors. We believe that, in this genomic era, computational methods can be applied to promote aquatic biodiversity conservation by addressing questions related to the evolutionary history of aquatic organisms at the molecular level. However, huge amounts of genomics data generated can only be discerned through the use of bioinformatics. Here, we examine the applications of next-generation sequencing technologies and bioinformatics tools to study the molecular evolution of aquatic animals and discuss the current challenges and future perspectives of using bioinformatics toward aquatic animal conservation efforts.

Hidden genetic diversity in snakeskin gourami, Trichopodus pectoralis (Perciformes, Osphronemidae), inferred from the mitochondrial DNA CO1 gene.

Eighty-four specimens collected from 13 populations from Malaysia, Thailand, and Vietnam were analysed, revealing 21 putative haplotypes with overall estimated haplotype and nucleotide diversities of 0.79 and 0.0079, respectively. High levels of diversity and an absence of founder effects were observed among populations in peninsular Malaysia. In contrast, populations from Sarawak exhibited low genetic diversity, which is a typical sign of colonies introduced from a single source. Historical translocation of Trichopodus pectoralis from Thailand to Malaysia, as well as to the Philippines, Indonesia, and Myanmar was apparent. Historical introduction of T. pectoralis from Vietnam was also detected in peninsular Malaysia.

The complete mitochondrial genome of the snakeskin gourami, Trichopodus pectoralis (Regan 1910) (Teleostei: Osphronemidae).

We sequenced and assembled three whole mitogenome sequences of the commercially important snakeskin gourami Trichopodus pectoralis isolated from Malaysia (introduced), Viet Nam (native) and Thailand (native). The mitogenome length range from 16,397 to 16,420 bp. The final partitioned nucleotide alignment consists of 14,002 bp and supports the monophyly of the genus Trichopodus (95% ultrafast bootstrap support) with T. trichopterus forming a sister group with the members of T. pectoralis.

Phylogeographic pattern of the striped snakehead, Channa striata in Sundaland: ancient river connectivity, geographical and anthropogenic signatures [corrected].

A phylogeographic study of an economically important freshwater fish, the striped snakehead, Channa striata in Sundaland was carried out using data from mtDNA ND5 gene target to elucidate genetic patterning. Templates obtained from a total of 280 individuals representing 24 sampling sites revealed 27 putative haplotypes. Three distinct genetic lineages were apparent; 1)northwest Peninsular Malaysia, 2)southern Peninsular, east Peninsular, Sumatra and SW (western Sarawak) and 3) central west Peninsular and Malaysian Borneo (except SW). Genetic structuring between lineages showed a significant signature of natural geographical barriers that have been acting as effective dividers between these populations. However, genetic propinquity between the SW and southern Peninsular and east Peninsular Malaysia populations was taken as evidence of ancient river connectivity between these regions during the Pleistocene epoch. Alternatively, close genetic relationship between central west Peninsular Malaysia and Malaysian Borneo populations implied anthropogenic activities. Further, haplotype sharing between the east Peninsular Malaysia and Sumatra populations revealed extraordinary migration ability of C. striata (>500 km) through ancient connectivity. These results provide interesting insights into the historical and contemporary landscape arrangement in shaping genetic patterns of freshwater species in Sundaland.